Our efforts to identify genes that regulate intestinal epithelial cell differentiation led to the discovery of Brk (Breast tumor kinase, also called PTK6 and Sik). Brk is a non-myristoylated, intracellular epithelial-specific tyrosine kinase that is expressed in breast tumors where it has been proposed to contribute to oncogenic signaling. However we discovered that Brk is expressed at highest levels in the normal intestine, where it is localized to nondividing, differentiated epithelial cells. To elucidate functions of Brk in vivo, we disrupted the mouse Brk gene, and found that loss of Brk enhanced growth and delayed enterocyte differentiation in the small intestine. In addition, we discovered that Brk is induced in intestinal epithelial crypt cells in response to ?-irradiation. We detected impaired apoptosis in the Brk-/- mouse after total body irradiation, indicating that induction of Brk in the crypts contributes to apoptosis. In addition our preliminary data suggest that Brk-/- mice are more susceptible to the colon carcinogen azoxymethane than wild type mice. We hypothesize that Brk regulates intestinal tissue homeostasis, and acts as a tumor suppressor in the intestinal tract, in contrast to its role in breast cancer. To define the roles of Brk in regulation of growth, differentiation, and apoptosis in intestinal epithelial cells, we propose: 1) To determine if Brk regulates growth and apoptosis by negatively regulating Akt using engineered cell lines, and wild type and Brk-deficient mice. In preliminary studies, we detected increased activation of Akt, a key positive regulator of growth and survival signaling and a substrate of Brk in intestines of Brk- /- mice;2) To explore mechanisms underlying the ability of Brk to regulate differentiation of intestinal epithelial cells, using novel cell culture and animal models, and 3) To evaluate contributions of Brk to tumorigenesis in the intestine. We will determine if Brk has tumor suppressor functions using a variety of different mouse colon cancer model systems and engineered cell lines. Our data suggest that Brk has different functions in normal intestine and in breast cancer, which may be dependent on its intracellular localization and access to different signaling molecules within the cell. Our studies are directed at determining the normal physiological functions of the Brk tyrosine kinase, which will be important for understanding its potential contributions to intestinal tissue homeostasis and the development of intestinal cancers.